Aminoglycoside antibiotics (AGAs) are a mainstay in clinical management of life-threatening Gram-negative bacterial infections. AGAs are widely available and highly economical;however, their use is limited due to ototoxicity and nephrotoxicity. We sought to identify agents that would block aminoglycoside ototoxicity without blocking its antimicrobial activity. Using an in vivo zebrafish (Danio rerio) neuromast assay, we identified two structurally related compounds (PROTO1 and 2) from a library of diverse, drug-like compounds that protect zebrafish mechanosensory neuromast cells from neomycin-induced cell death. The protective effect of PROTO1/2 is dose-dependent and is not due to inhibition of AGA uptake into zebrafish neuromasts. Furthermore, neither PROTO1 nor PROTO2 inhibit AGA antibacterial activity in E. coli. PROTO1 is non-toxic in animals and confers AGA resistance in explanted mouse utricles and human kidney cells. In this application we propose four Specific Aims to carry out pre-clinical optimization of the lead compounds, to identify the molecular targets of the protective drugs and determine the molecular pathways that are involved in both AGA-induced ototoxicity and its prevention by PROTO1/2. Specifically, in Aim 1, we will synthesize and evaluate the hair cell protective effects of PROTO1/2 analogues with two goals: 1). to increase the potency of the protective activity, and;2). to develop a structure activity relationship (SAR) that would allow us to modify PROTO1/2 for target identification studies (Aim 3). In Aim 2, we will develop AGA-induced toxicity assays using human kidney cells, zebrafish neuromast hair cells and explanted mouse utricles. In Aim 3, we will carry out in vivo pre-clinical evaluation of PROTO1/2-derived agents. In Aim 4, we will use SAR data to design and synthesize an affinity capture reagent based on PROTO1/2 and will carry out target identification studies. These convergent and complementary studies will aid in the development of target-based hair cell protective agents and expand the molecular understanding of AGA-induced cell damage. The use of many drugs is limited by serious side effects. We have discovered compounds that may help reduce hearing loss and damage to kidneys caused by an important class of antibiotics. Studies in this proposal will develop more potent protective compounds, identify how these compounds work and provide preliminary data for their development as drugs.